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The Clinical and Radiological Spectrum of Hippocampal Pathology in Amyotrophic Lateral Sclerosis

Hippocampal pathology in Amyotrophic Lateral Sclerosis (ALS) remains surprisingly under recognized despite compelling evidence from neuropsychology, neuroimaging and neuropathology studies. Hippocampal dysfunction contributes significantly to the clinical heterogeneity of ALS and requires structure-specific cognitive and neuroimaging tools for accurate in vivo evaluation. Recent imaging studies have generated unprecedented insights into the presymptomatic and longitudinal processes affecting this structure and have contributed to the characterisation of both focal and network-level changes. Emerging neuropsychology data suggest that memory deficits in ALS may be independent from executive dysfunction. In the era of precision medicine, where the development of individualized care strategies and patient stratification for clinical trials are key priorities, the comprehensive review of hippocampal dysfunction in ALS is particularly timely.


INTRODUCTION
Amyotrophic lateral sclerosis (ALS) is relentlessly progressive neurodegenerative condition with considerable clinical heterogeneity (1). One of the key clinical dimensions of disease heterogeneity in ALS is the varying severity and profile of cognitive impairment. The quality of life implications of cognitive impairment in ALS and its impact on caregiver burden (2), compliance with assistive devices (3) and survival (4) are now universally recognized. The discovery of hexanucleotide expansions in C9orf72 in 2011 (5) has given fresh momentum to neuropsychology research in ALS by confirming shared etiological factors between frontotemporal dementia (FTD) and ALS. The momentous conceptual advances in the neuropsychology of ALS have taken place in a remarkably short period of time, from sporadic observations, through the development of diagnostic criteria (6), to robust family aggregation (7) and genetic studies, to the development of disease-specific screening instruments (8,9). The current consensus criteria (6) distinguish ALS with cognitive impairment; ALS with behavioral impairment; ALS with cognitive and behavioral impairment; ALS-FTD; ALS-dementia (non-FTD, i.e., Alzheimer dementia (AD), vascular dementia, mixed dementia). One of the most exciting aspects of ALS neuropsychology studies is their localization potential to specific anatomical circuits and that their observations are widely corroborated by neuropathology (10)(11)(12) and neuroimaging studies (13). Memory deficits in ALS have traditionally been regarded as atypical and considered suggestive of coexisting AD-type pathology. The recognition that memory deficits are part of the spectrum of ALS-associated cognitive impairment is relatively recent.
II of the entorhinal cortex, its axons perforate the subiculum, and form the "perforant pathway" (PP). Duvernoy (24) coined the term "polysynaptic pathway" for the intrinsic hippocampal circuitry, which refers to a circuit of at least four synapses that connect the entorhinal cortex, the dentate gyrus, the CA subfields, and the subiculum. A direct intrahippocampal pathway has also been identified, which originates from layer III of the entorhinal cortex and projects directly to the CA1 but not through the PP (25). The perforant pathway ( Figure 1C) arises from layer II-III neurons of the lateral and medial entorhinal cortex (26), which is also the origin of the polysynaptic pathway (27). The PP perforates the subiculum to reach the dentate gyrus and the hippocampus proper, but minor projections also originate from the presubiculum and parasubiculum (28). The majority of the PP fibers reach the stratum moleculare of the dentate gyrus through the vestigial hippocampal sulcus (24). The PP contributes to the "Papez circuit" (26,29,30) which is relayed through the following structures; entorhinal cortex → dentate gyrus → hippocampus → hypothalamus → thalamus → cingulate cortex→ presubiculum → entorhinal cortex. In addition to the intrinsic hippocampal circuitry, there are numerous extrinsic hippocampal projections to subcortical and cortical regions (31). The main input to the hippocampus enters via the entorhinal area (31).

INSIGHTS FROM NEUROPATHOLOGY
Neuropathological changes have been consistently reported in the hippocampus in ALS ( Table 1). Early reports highlighted ubiquitin-positive intraneuronal inclusions (32)(33)(34)(35) in medial temporal structures, neuronal loss in the medial cortex of the temporal tip (36,69) and focal depletion of pyramidal neurons in the pes hippocampi in both patients with and without dementia (33,36,69). A specific focus of interest in histopathological studies is the PP zone, which has been comprehensively studied in most neurodegenerative conditions, especially in AD. While AD is characterized by the extraneuronal deposits of the amyloid β-protein (Aβ) and the intraneuronal tauopathy (70), ALS is primarily associated with TAR DNA-binding protein 43 (TDP-43) deposits (71). ALS patients with and without dementia (37,38) show neuropathological changes in the dentate gyrus and the outer lamina of the molecular layer where the PP terminals are distributed (26,30,72). In ALS, the molecular layer of the dentate gyrus is primarily affected, a pattern which is distinctly different from AD. The inner molecular layer, which is innervated by the CA4 (73), is the least affected layer in ALS (38). The intermediate layer, which receives projections from layer II of the medial entorhinal cortex, is affected (38), but the outer layer, is the most affected region in ALS (38). Despite considerable mesial temporal lobe involvement in both ALS and AD, the distribution of disease-specific inclusions is strikingly different. Neurofibrillary tangles in AD are mostly found in the entorhinal cortex and are positive for tau, whereas the main proteinopathy of ALS is TDP-43 and mostly affects the transentorhinal cortex (38).
It is now widely recognized that phosphorylated TDP-43 (pTDP-43) aggregates are the hallmark pathology of sporadic ALS (39,74,75). Based on post mortem observations, a sequential staging system of pTDP-43 pathology has been proposed, using stage-defining involvement of specific cortical and subcortical regions (12). According to this four-stage model of disease propagation, the PP is predominantly affected in stage IV. A three-stage model has also been suggested for PP degeneration (38) where stage I is the "inclusion stage" defined by TDP-43-positive cytoplasmatic inclusions appearing in the granular cells of the dentate gyrus, stage II is the "early perforant stage" where gliosis and neuronal loss of the transentorhinal cortex are observed, and stage III is the "advanced perforant stage" defined by the degeneration of the molecular layer of the dentate gyrus and neuronal loss in the transentorhinal cortex (38). The chronological stages of hippocampal pathology are closely linked to its structural anatomy, confirming that disease propagation occurs along connectivity patterns (76). The TDP-43 stages of ALS are in line with the notion that gray matter (GM) regions become sequentially involved via the WM pathways that connect them (77)(78)(79).

THE CONTRIBUTION OF NEUROIMAGING
Neuroimaging studies have already contributed meaningful structural, metabolic and functional insights in ALS (80,81) and recent technological advances in imaging techniques offer unprecedented opportunities to characterize hippocampal changes in vivo. Following sporadic reports of hippocampal degeneration (82)(83)(84)(85) in whole-brain exploratory studies, recent studies have specifically focused on the evaluation of this structure (43) ( Table 1). Emerging imaging methods not only highlight hippocampal volume reductions, but have the potential to characterize specific sub-regions (78), shape changes (42), density alterations (20), progressive longitudinal changes (43), altered connectivity profiles, and functional changes (40,46,47).

Structural Neuroimaging
Computational neuroimaging techniques have consistently captured hippocampal GM changes which was initially thought to be more significant in ALS patients carrying the C9orf72 hexanucleotide repeats (40), but later studies showed similarly extensive hippocampal degeneration in C9orf72 negative ALS-FTD patients (78). Interestingly, unilateral hippocampal changes were not only captured in patients with cognitive impairment (42), but also in cognitively intact cohorts (41). Shape and density analyses of the hippocampus in ALS highlighted phenotype-specific patterns of hippocampal degeneration (42). A longitudinal study of hippocampus, which included a small (∼6%) number of C9orf72 positive patients, identified baseline  changes in the left presubiculum, and progressive CA2/3, CA4 and the left presubiculum involvement at follow-up (43). While diffusion-weighted imaging (DWI) is primarily used to study white matter (WM) structures, there is increasing evidence that it may provide useful information on aspects of GM integrity (86). Evaluation of diffusion tensor imaging (DTI) metrics have consistently shown low fractional anisotropy (44,49) and increased mean diffusivity in both hippocampal (44)(45)(46)(47) and parahippocampal regions (48).
DTI has been initially used to characterize medial temporal lobe WM regions and later to assess limbic circuit integrity (i.e., fornix; uncinate fasciculus) (87)(88)(89). One of the most unique applications of hippocampal DTI in ALS however is the ability to reconstruct and evaluate of the PP. (50,51). Based on in vivo assessments, these studies have not only confirmed previous neuropathological observations but also revealed structure-specific clinical correlations (51). The use of DWI-based PP imaging (90) has contributed to our understanding of impaired memory processing in a range of conditions from mild cognitive impairment, through AD, to traumatic brain injury (91)(92)(93)(94). PP imaging is therefore a relatively well-established approach which has only recently been applied to ALS. A longitudinal tractography study of ALS (52) found increased connectivity between the visual cortex and medial temporal lobe regions which increased further at 3-month follow-up. Increased connectivity over time in ALS is not an isolated finding (95) and is often interpreted as a compensatory mechanism.

Functional Neuroimaging
There are relatively few paradigm-based functional magnetic resonance imaging (fMRI) studies specifically evaluating hippocampal function, but a longitudinal fMRI study identified increased novelty-evoked hippocampal activity over time (53). Resting-state studies have consistently captured increased connectivity between the left sensorimotor cortex and contralateral cortical regions including the parahippocampal gyrus (54). Additionally, increased low-frequency amplitudes have been observed in the right parahippocampal cortex (55). Increased functional connectivity was also identified between parahippocampal components of the default-mode network (56). In a relatively large sample of ALS patients with only minor cognitive changes, (57) decreased functional connectivity was identified between temporal lobe structures, including hippocampal and parahippocampal regions. This was thought to represent early metabolic disturbances before cell-loss occurs but highlight the fact that increased and decreased connectivity is both reported in fMRI studies of ALS.

INSIGHTS FROM NEUROPSYCHOLOGY
Contrary to the consensus around executive dysfunction in ALS (96)(97)(98)(99), there are strikingly inconsistent reports about the incidence of memory impairment in ALS ( Table 1). Intact memory function, mild dysfunction, executive function mediated memory impairment, and moderate memory deficits have all been reported (58)(59)(60)(61)(62)(63)(64)(65)97). The primary substrate of amnestic deficits is still under investigation. Most studies agree that the primary deficit is in encoding-retrieval (65) which is often linked to frontal dysfunction, attention, and other executive-based processes (65)(66)(67)(68). However, recognition deficits and memory consolidation difficulties are likely to be just as important (66). Compelling evidence also exist for pure episodic memory dysfunction based on impaired picture recall, word list-learning, pair associations, and story-recall. These observations would suggest that memory impairment in non-demented ALS patients cannot be exclusively attributed to executive dysfunction (100)(101)(102).
In a combined neuroimaging-neuropsychology study, abnormal immediate and delayed recall scores were identified in 23% of non-demented ALS patients (102). While the ALS cohort of this study did not exhibit reduced hippocampal volumes in comparison to healthy controls, their memory performance correlated with hippocampal volumes. These findings are echoed by other studies which rely on volumetric analyses and verbal list-learning test and report significant correlations between the hippocampal volumes and verbal memory indices such as total learning, delayed recall, and recognition (41).
While direct clinico-radiological correlations are often regarded as contentious (103), a positive association has been reported between verbal memory indices and hippocampal volumes in several ALS subgroups, including ALSci and ALS-FTD (42). DTI studies have consistently revealed correlations between memory performance and memory-associated WM tracts such as the fornix (88), the uncinate fasciculus (87,88), and the hippocampal PP (51). Emerging reports of similar episodic memory performance in ALS and amnestic mild cognitive impairment patients (67) corroborates neuropathological findings of comparable PP changes (37,38).

Testing Recommendations
Traditionally, the assessment of episodic memory includes tests for immediate and delayed recall, and performance evaluated from a learning, retention and recognition perspective. More recently, distinct memory processes are specifically assessed, such as encoding, consolidation, and retrieval. (104-106) Listlearning tests (e.g., California Verbal Learning Test; Rey Auditory Verbal Learning Test; Hopkins Verbal Learning Test etc.) are particularly useful to assess hippocampus-mediated verbal memory dysfunction in ALS. These tests enable the clinician to evaluate immediate recall, delayed recall, and recognition and can be readily interpreted in terms of encoding, consolidation, and retrieval performance (66). Story-recall tests, such as the Wechsler-Memory Scale, are also sensitive to detect episodic memory impairment and ideally, both list-learning and storyrecall should be performed to comprehensively evaluate episodic memory in ALS. The accurate assessment of visual episodic memory is often confounded by motor disability in in ALS or by coexisting executive dysfunction which may affect the organization and encoding of complex figures (e.g., Rey-Osterreith Complex Figure Test). The limitations of short, non-ALS, cognitive screening tools such as MMSE; ACE; MoCA are widely recognized in the ALS research community, as these tests have been developed for other neurodegenerative conditions. The administration of ALS specific screening tools (ECAS, ALS-CBS) should be followed by specialist neuropsychological evaluation if memory impairment is identified or reported by the patient or caregiver.

DISCUSSION
The synthesis of insights from neuropathology, neuroimaging and neuropsychology enables the systematic discussion of structural and functional aspects of hippocampal degeneration in ALS and helps to integrate focal pathology into a network perspective.
While hippocampal pathology used to be primarily evaluated in ALS patients with comorbid dementia (34,37,38), recent studies have increasingly focused on non-demented patient cohorts (12,32,69,71). With the increased recognition of neuropsychological deficits beyond executive dysfunction, imaging studies of ALS have gradually started to evaluate mesial temporal lobe structures and memory domains have now been incorporated in ALS-specific cognitive screening tools (8). The targeted evaluation of memory function and reliance on more sophisticated indices of episodic memory (65-68) not only help to characterize the heterogeneity of cognitive profiles but also confirm that pure episodic memory dysfunction is not uncommon in ALS and can be detected in the absence of FTD.
Despite the momentous advances in characterizing hippocampal degeneration in ALS, considerable shortcomings and inconsistencies can be identified. The commonest problem is sample size limitations followed by the inclusion of poorly characterized patients. The comprehensive neuropsychological assessment of patients is paramount and administering screening tests alone is not sufficient. Reliance on non-ALS specific batteries, such as Addenbrooke's Cognitive Examination, Mini-Mental State Examination, Montreal Cognitive Assessment, is not sufficient to characterize ALS-associated cognitive change. A common shortcoming of ALS neuropsychology papers is overlooking the confounding effect of medications which affect cognitive performance. Anticholinergics commonly used for sialorrhea, tricyclic antidepressants, opiates, benzodiazepines are all widely used in ALS and have a significant impact on attention, registration, and recall. Other disease-specific confounding factors such as hypoxia, hypercapnia, physical discomfort, fatigue, apathy, low mood, depression also need careful consideration. Despite established consensus criteria (6) different batteries are used in different centers to test memory. There is a paucity of reports where caregivers or family members are interviewed about the sort of memory impairment they may have observed. A few targeted questions if the patient gets lost in familiar places, misplaces items, forgets names, or dates etc. may be worth asking from the caregivers. Given the strikingly quick progression rates observed in ALS compared to other neurodegenerative conditions, resource allocation, care planning, assessment of capacity may be important at an early stage of the disease. ALS patients have to make a number of important financial, personal, and end-of-life decisions which may or may not be affected by memory impairment.
The practice of excluding patients with dementia in neuroimaging studies (44,47,49,55) to evaluate clinically homogenous samples may also be counterintuitive. More recent imaging papers include comprehensive cognitive testing (55)(56)(57) which aids the interpretation of extra-motor changes (107). The lack of cognitive profiling of the healthy controls in many neuroimaging studies also precludes robust statistics as only the patient group is then used for correlative analyses. Often, reference normative neuropsychology data are used for the interpretation of patient's memory performance, data which is independent from the given study and originate from volunteers who have not been scanned as part of the given study. The patients' neuroimaging data on the other hand are contrasted to scans of controls who had no detailed neuropsychological evaluation. This unfortunately is a common study design, which essentially uses a different imaging and neuropsychology control group. Another common shortcoming of ALS neuroimaging studies is the lack of adjustment for education, which may impact on both structural and functional imaging data (80). A binary, comparative study design of patients versus controls and the contrasting of two clinically or genetically defined cohorts is not entirely satisfactory either. The inclusion of mimic cohorts, or a "disease-control" group with an alternative neurodegenerative condition such as MCI, AD, or Parkinson disease would also be desirable. The selection bias of relatively well patients who are able to lie flat in the scanner and able to make the journey to a radiology department is seldom acknowledged. It is conceivable that progressive hippocampal changes occur as the disease progresses, but these patients are no longer able to partake in imaging studies. Clinical trial designs are not only hampered by late recruitment of clinically heterogeneous cohorts, but they overwhelmingly rely on motor, respiratory, nutritional markers (108)(109)(110). Patient stratification based on cognitive performance prior to inclusion and monitoring performance during the trial seems essential, especially given the survival implications of cognitive impairment (3,4,111).
Despite initial enthusiasm that hexanucleotide repeats account for most of the ALS-FTD cohort (112,113), it has quickly become apparent that C9orf72 hexanucleotide repeats only explain a minority of ALS-FTD cases (114). Emerging studies confirm that a subgroup of C9orf72 negative patients may show neuroanatomical alterations similar to the ones observed in patients carrying the hexanucleotide expansion. Furthermore, temporal lobe changes have been captured in asymptomatic hexanucleotide carriers, who also exhibited subcortical gray matter degeneration prior to symptom onset (115).
Existing multimodal studies which combine neuroimaging and neurocognitive measures either support a close association between anatomical changes and memory performance or highlight a relative dissociation between the two methods. This inconsistency is epitomized by reports of absent neuroimaging changes in patients with established memory deficits and the detection of significant hippocampal changes in patients with mild memory impairment (41,42,102). Based on the shortcomings of existing hippocampal studies in ALS, future studies should include large sample sizes, disease-controls, longitudinal designs, paradigm-based fMRI, comprehensive neuropsychological profiling, "disease-controls, " anatomical corrections for education, and genetic screening for mutations implicated in ALS, FTD, and AD. Furthermore, reliance on high directional diffusion models such as neurite orientation dispersion and density imaging (NODDI), high angular resolution diffusion imaging (HARDI), or Q-ball imaging may be desirable to characterize early WM alterations in parahippocampal regions. Finally, combined imaging and postmortem studies may provide a validation of the in vivo findings.
In conclusion, hippocampal pathology is a clinically and academically relevant field of ALS research which has gained unprecedented momentum in recent years and is likely to contribute important further insights in the coming years.

AUTHOR CONTRIBUTIONS
The paper was drafted by FC, EK, and PB and has been reviewed for intellectual content by GV, PF, MR, NK, and IE.