Distinct α-synuclein strains differentially promote tau inclusions in neurons.

Many neurodegenerative diseases are characterized by the accumulation of insoluble protein aggregates, including neurofibrillary tangles comprised of tau in Alzheimer's disease and Lewy bodies composed of α-synuclein in Parkinson's disease. Moreover, different pathological proteins frequently codeposit in disease brains. To test whether aggregated α-synuclein can directly cross-seed tau fibrillization, we administered preformed α-synuclein fibrils assembled from recombinant protein to primary neurons and transgenic mice. Remarkably, we discovered two distinct strains of synthetic α-synuclein fibrils that demonstrated striking differences in the efficiency of cross-seeding tau aggregation, both in neuron cultures and in vivo. Proteinase K digestion revealed conformational differences between the two synthetic α-synuclein strains and also between sarkosyl-insoluble α-synuclein extracted from two subgroups of Parkinson's disease brains. We speculate that distinct strains of pathological α-synuclein likely exist in neurodegenerative disease brains and may underlie the tremendous heterogeneity of synucleinopathies.

Selective Motor Neuron Resistance and Recovery in a New Inducible Mouse Model of TDP-43 Proteinopathy.

UNLABELLED: Motor neurons (MNs) are the neuronal class that is principally affected in amyotrophic lateral sclerosis (ALS), but it is widely known that individual motor pools do not succumb to degeneration simultaneously. Because >90% of ALS patients have an accumulation of cytoplasmic TDP-43 aggregates in postmortem brain and spinal cord (SC), it has been suggested that these inclusions in a given population may trigger its death. We investigated seven MN pools in our new inducible rNLS8 transgenic (Tg) mouse model of TDP-43 proteinopathy and found striking differences in MN responses to TDP-43 pathology. Despite widespread neuronal expression of cytoplasmic human TDP-43, only MNs in the hypoglossal nucleus and the SC are lost after 8 weeks of transgene expression, whereas those in the oculomotor, trigeminal, and facial nuclei are spared. Within the SC, slow MNs survive to end stage, whereas fast fatigable MNs are lost. Correspondingly, axonal dieback occurs first from fast-twitch muscle fibers, whereas slow-twitch fibers remain innervated. Individual pools show differences in the downregulation of endogenous nuclear TDP-43, but this does not fully account for vulnerability to degenerate. After transgene suppression, resistant MNs sprout collaterals to reinnervate previously denervated neuromuscular junctions concurrently with expression of matrix metalloproteinase 9 (MMP-9), a marker of fast MNs. Therefore, although pathological TDP-43 is linked to MN degeneration, the process is not stochastic and mirrors the highly selective patterns of MN degeneration observed in ALS patients. SIGNIFICANCE STATEMENT: Because TDP-43 is the major pathological hallmark of amyotrophic lateral sclerosis (ALS), we generated mice in which mutant human TDP-43 expression causes progressive neuron loss. We show that these rNLS8 mice have a pattern of axonal dieback and cell death that mirrors that often observed in human patients. This finding demonstrates the diversity of motor neuron (MN) populations in their response to pathological TDP-43. Furthermore, we demonstrate that resistant MNs are able to compensate for the loss of their more vulnerable counterparts and change their phenotype in the process. These findings are important because using a mouse model that closely models human ALS in both the disease pathology and the pattern of degeneration is critical to studying and eventually treating progressive paralysis in ALS patients.

An insoluble frontotemporal lobar degeneration-associated TDP-43 C-terminal fragment causes neurodegeneration and hippocampus pathology in transgenic mice.

Frontotemporal dementia (FTD) causes progressive personality, behavior and/or language disturbances and represents the second most common form of dementia under the age of 65. Over half of all FTD cases are classified pathologically as frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein of 43 kDa (TDP-43) pathology (FTLD-TDP). In FTLD-TDP brains, TDP-43 is phosphorylated, C-terminally cleaved, lost from the nucleus and accumulates in the cytoplasm and processes of neurons and glia. However, the contribution of TDP-43 C-terminal fragments (CTFs) to pathogenesis remains poorly understood. Here, we developed transgenic (Tg) mice with forebrain Camk2a-controlled doxycycline-suppressible expression of a TDP-43 CTF (amino acids 208-414, designated 208 TDP-43 CTF), previously identified in FTLD-TDP brains. In these 208 TDP-43 Tg mice, detergent-insoluble 208 TDP-43 CTF was present in a diffuse punctate pattern in neuronal cytoplasm and dendrites without forming large cytoplasmic inclusions. Remarkably, the hippocampus showed progressive neuron loss and astrogliosis in the dentate gyrus (DG). This was accompanied by phosphorylated TDP-43 in the CA1 subfield, and ubiquitin and mitochondria accumulations in the stratum lacunosum moleculare (SLM) layer, without loss of endogenous nuclear TDP-43. Importantly, 208 TDP-43 CTF and phosphorylated TDP-43 were rapidly cleared when CTF expression was suppressed in aged Tg mice, which ameliorated neuron loss in the DG despite persistence of ubiquitin accumulation in the SLM. Our results demonstrate that Camk2a-directed 208 TDP-43 CTF overexpression is sufficient to cause hippocampal pathology and neurodegeneration in vivo, suggesting an active role for TDP-43 CTFs in the pathogenesis of FTLD-TDP and related TDP-43 proteinopathies.

Expansion of the classification of FTLD-TDP: distinct pathology associated with rapidly progressive frontotemporal degeneration.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) can typically be categorized into one of four distinct histopathologic patterns of TDP-43 pathology, types A to D. The strength of this histopathologic classification lies in the association between FTLD-TDP subtypes and various clinical and genetic features of disease. Seven cases of FTLD-TDP were identified here which were difficult to classify based on existing pathologic criteria. Distinct features common to these cases included TDP-43 aggregates over a wide neuroanatomic distribution comprised of granulofilamentous neuronal inclusions, abundant grains, and oligodendroglial inclusions. TDP-43 aggregates were phosphorylated and associated with loss of normal nuclear TDP-43 protein (nuclear clearance) but were negative for ubiquitin. Biochemical analysis confirmed the presence of insoluble and phosphorylated TDP-43 and also revealed a distinct pattern of TDP-43 C-terminal fragments relative to other FTLD-TDP subtypes. Finally, these cases were uniformly associated with a very rapid clinical course culminating in death within ~3 years of disease onset. We suggest that these cases may represent a unique clinicopathologic subtype of FTLD-TDP which we provisionally call "type E." The immature appearance of TDP-43 aggregates, widespread distribution, uniform biochemical profile and rapid clinical course highlights the clinical and pathologic variability within FTLD-TDP, and raises the possibility that type E neuropathology is the sequelae of a particularly virulent strain of TDP-43 proteinopathy.

Functional recovery in new mouse models of ALS/FTLD after clearance of pathological cytoplasmic TDP-43.

Accumulation of phosphorylated cytoplasmic TDP-43 inclusions accompanied by loss of normal nuclear TDP-43 in neurons and glia of the brain and spinal cord are the molecular hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the role of cytoplasmic TDP-43 in the pathogenesis of these neurodegenerative TDP-43 proteinopathies remains unclear, due in part to a lack of valid mouse models. We therefore generated new mice with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (∆NLS) under the control of the neurofilament heavy chain promoter. Expression of hTDP-43∆NLS in these 'regulatable NLS' (rNLS) mice resulted in the accumulation of insoluble, phosphorylated cytoplasmic TDP-43 in brain and spinal cord, loss of endogenous nuclear mouse TDP-43 (mTDP-43), brain atrophy, muscle denervation, dramatic motor neuron loss, and progressive motor impairments leading to death. Notably, suppression of hTDP-43∆NLS expression by return of Dox to rNLS mice after disease onset caused a dramatic decrease in phosphorylated TDP-43 pathology, an increase in nuclear mTDP-43 to control levels, and the prevention of further motor neuron loss. rNLS mice back on Dox also showed a significant increase in muscle innervation, a rescue of motor impairments, and a dramatic extension of lifespan. Thus, the rNLS mice are new TDP-43 mouse models that delineate the timeline of pathology development, muscle denervation and neuron loss in ALS/FTLD-TDP. Importantly, even after neurodegeneration and onset of motor dysfunction, removal of cytoplasmic TDP-43 and the concomitant return of nuclear TDP-43 led to neuron preservation, muscle re-innervation and functional recovery.

TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/212 cluster and affects progranulin pathways.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a fatal neurodegenerative disease with no available treatments. Mutations in the progranulin gene (GRN) causing impaired production or secretion of progranulin are a common Mendelian cause of FTLD-TDP; additionally, common variants at chromosome 7p21 in the uncharacterized gene TMEM106B were recently linked by genome-wide association to FTLD-TDP with and without GRN mutations. Here we show that TMEM106B is neuronally expressed in postmortem human brain tissue, and that expression levels are increased in FTLD-TDP brain. Furthermore, using an unbiased, microarray-based screen of >800 microRNAs (miRs), we identify microRNA-132 as the top microRNA differentiating FTLD-TDP and control brains, with <50% normal expression levels of three members of the microRNA-132 cluster (microRNA-132, microRNA-132*, and microRNA-212) in disease. Computational analyses, corroborated empirically, demonstrate that the top mRNA target of both microRNA-132 and microRNA-212 is TMEM106B; both microRNAs repress TMEM106B expression through shared microRNA-132/212 binding sites in the TMEM106B 3'UTR. Increasing TMEM106B expression to model disease results in enlargement and poor acidification of endo-lysosomes, as well as impairment of mannose-6-phosphate-receptor trafficking. Finally, endogenous neuronal TMEM106B colocalizes with progranulin in late endo-lysosomes, and TMEM106B overexpression increases intracellular levels of progranulin. Thus, TMEM106B is an FTLD-TDP risk gene, with microRNA-132/212 depression as an event which can lead to aberrant overexpression of TMEM106B, which in turn alters progranulin pathways. Evidence for this pathogenic cascade includes the striking convergence of two independent, genomic-scale screens on a microRNA:mRNA regulatory pair. Our findings open novel directions for elucidating miR-based therapies in FTLD-TDP.

TDP-43 skeins show properties of amyloid in a subset of ALS cases.

Aggregation of TDP-43 proteins to form intracellular inclusions is the primary pathology in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 inclusions (FTLD-TDP). Histologically, in the cerebral cortex and limbic regions of affected ALS and FTLD-TDP patients, these pathologies occur as a variety of cytoplasmic, neuritic and intranuclear TDP-43 inclusions. In the spinal cord and lower brainstem of ALS patients, the lesions form cytoplasmic dashes or complex filamentous and spherical profiles in addition to skein-like inclusions (SLI). Ultrastructurally, the morphology of TDP-43 inclusions is heterogeneous but mainly composed of loose bundles of 10- to 20-nm-diameter straight filaments associated with electron-dense granular material. All of these TDP-43 inclusions are generally described as disordered amorphous aggregations unlike the amyloid fibrils that characterize protein accumulations in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We here report that Thioflavin-S positive SLI are present in a subset of ALS cases, while TDP-43 inclusions outside the spinal cord lack the chemical properties of amyloid. Further, we examine the differential enrichment of fibrillar profiles in SLI of ALS cases by TDP-43 immuno-electron microscopy (immuno-EM). The demonstration that pathological TDP-43 can be amyloidogenic in situ suggests the following conclusions: (1) the conformational changes associated with TDP-43 aggregation are more complex than previously thought; (2) Thioflavin-S positive SLI may be composed primarily of filamentous ultrastructures.

Motor neuron disease clinically limited to the lower motor neuron is a diffuse TDP-43 proteinopathy.

Motor neuron disease (MND) may present as an isolated lower motor neuron (LMN) disorder. Although the significance of pathological 43 kDa transactive responsive sequence DNA binding protein (TDP-43) for amyotrophic lateral sclerosis (ALS) was appreciated only recently, the topographical distribution of TDP-43 pathology in MND clinically isolated to the LMN versus normal controls (COs) is only incompletely described. Therefore, we performed longitudinal clinical evaluation and retrospective chart review of autopsied patients diagnosed with isolated LMN disease. Cases with a disease duration over 4 years were designated as progressive muscular atrophy (PMA), and those with a more rapid course as MND/LMN. Immunohistochemistry was employed to identify neuronal and glial TDP-43 pathology in the central nervous system (CNS) in patients and COs. We examined 19 subjects including six patients (i.e., four with MND/LMN and two with PMA) and 13 COs. All patients showed significant TDP-43 linked degeneration of LMNs, and five cases showed a lesser degree of motor cortex degeneration. Additional brain areas were affected in varying degrees, ranging from predominantly brainstem pathology to significant involvement of the whole CNS including neocortical and limbic areas. Pathological TDP-43 was present only rarely in the CO group. We conclude that MND limited to the LMN and PMA is part of a disease continuum that includes ALS and FTLD-TDP, all of which are characterized by widespread TDP-43 pathology. Hence, we suggest that the next revision of the El Escorial criteria for the diagnosis of ALS include MND patients with disease clinically limited to the LMN and PMA as variants of ALS, which like classical ALS, are TDP-43 proteinopathies.

Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration is a fatal neurodegenerative disease that results in progressive decline in behavior, executive function and sometimes language. Disease mechanisms remain poorly understood. Recently, however, the DNA- and RNA-binding protein TDP-43 has been identified as the major protein present in the hallmark inclusion bodies of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U), suggesting a role for transcriptional dysregulation in FTLD-U pathophysiology. Using the Affymetrix U133A microarray platform, we profiled global gene expression in both histopathologically affected and unaffected areas of human FTLD-U brains. We then characterized differential gene expression with biological pathway analyses, cluster and principal component analyses, and subgroup analyses based on brain region and progranulin (GRN) gene status. Comparing 17 FTLD-U brains to 11 controls, we identified 414 upregulated and 210 downregulated genes in frontal cortex (P-value < 0.001). Moreover, cluster and principal component analyses revealed that samples with mutations or possibly pathogenic variations in the GRN gene (GRN+, 7/17) had an expression signature that was distinct from both normal controls and FTLD-U samples lacking GRN gene variations (GRN-, 10/17). Within the subgroup of GRN+ FTLD-U, we found >1300 dysregulated genes in frontal cortex (P-value < 0.001), many participating in pathways uniquely dysregulated in the GRN+ cases. Our findings demonstrate a distinct molecular phenotype for GRN+ FTLD-U, not readily apparent on clinical or histopathological examination, suggesting distinct pathophysiological mechanisms for GRN+ and GRN- subtypes of FTLD-U. In addition, these data from a large number of human brains provide a valuable resource for future testing of disease hypotheses.

Enrichment of C-terminal fragments in TAR DNA-binding protein-43 cytoplasmic inclusions in brain but not in spinal cord of frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.

Phosphorylation of S409/410 of TDP-43 is a consistent feature in all sporadic and familial forms of TDP-43 proteinopathies.

Accumulation of hyperphosphorylated, ubiquitinated and N-terminally truncated TAR DNA-binding protein (TDP-43) is the pathological hallmark lesion in most familial and sporadic forms of FTLD-U and ALS, which can be subsumed as TDP-43 proteinopathies. In order to get more insight into the role of abnormal phosphorylation in the disease process, the identification of specific phosphorylation sites and the generation of phosphorylation-specific antibodies are mandatory. Here, we developed and characterized novel rat monoclonal antibodies (1D3 and 7A9) raised against phosphorylated S409/410 of TDP-43. These antibodies were used to study the presence of S409/410 phosphorylation by immunohistochemistry and biochemical analysis in a large series of 64 FTLD-U cases with or without motor neuron disease including familial cases with mutations in progranulin (n = 5), valosin-containing protein (n = 4) and linkage to chromosome 9p (n = 4), 18 ALS cases as well as other neurodegenerative diseases with concomitant TDP-43 pathology (n = 5). Our data demonstrate that phosphorylation of S409/410 of TDP-43 is a highly consistent feature in pathologic inclusions in the whole spectrum of sporadic and familial forms of TDP-43 proteinopathies. Physiological nuclear TDP-43 was not detectable with these mAbs by immunohistochemistry and by immunoblot analyses. While the accumulation of phosphorylated C-terminal fragments was a robust finding in the cortical brain regions of FTLD-U and ALS, usually being much more abundant than the phosphorylated full-length TDP-43 band, spinal cord samples revealed a predominance of full-length TDP-43 over C-terminal fragments. This argues for a distinct TDP-43 species composition in inclusions in cortical versus spinal cord cells. Overall, these mAbs are powerful tools for the highly specific detection of disease-associated abnormal TDP-43 species and will be extremely useful for the neuropathological routine diagnostics of TDP-43 proteinopathies and for the investigation of emerging cellular and animal models for TDP-43 proteinopathies.

Concomitant TAR-DNA-binding protein 43 pathology is present in Alzheimer disease and corticobasal degeneration but not in other tauopathies.

Pathologic TAR-DNA-binding protein 43 (TDP-43) is a disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. We studied the presence, frequency, and distribution of TDP-43 pathology by immunohistochemistry and biochemistry in a series of clinically well-characterized tauopathy patient brains, including 182 Alzheimer disease (AD), 39 corticobasal degeneration, 77 progressive supranuclear palsy, and 12 Pick disease cases and investigated the clinical impact of concomitant TDP-43 pathology in these cases. TAR-DNA-binding protein 43 pathology was found in 25.8% of AD cases. It was restricted to the dentate gyrus and entorhinal cortex in approximately 75% of cases; approximately 25% showed more widespread TDP-43 pathology in frontal and temporal cortices, resembling the FTLD-U subtype associated with progranulin mutations. TAR-DNA-binding protein 43 pathology in AD was associated with significantly longer disease duration, but there was no association with the clinical presentation (148 cases diagnosed as AD and 34 cases diagnosed as frontotemporal lobar degeneration). Progressive supranuclear palsy and Pick disease cases showed no TDP-43 inclusions and no biochemical alterations of TDP-43. There was, however, a unique, predominantly glial TDP-43 pathology with staining of astrocytic plaque-like structures and coiled bodies in 15.4% of corticobasal degeneration cases; this was associated with biochemical TDP-43 changes similar to those in FTLD-U. These findings provide further insight into the burden and clinical significance of TDP-43 pathology in disorders other than FTLD-U and amyotrophic lateral sclerosis.

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro.

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.

Tauopathy with hippocampal 4-repeat tau immunoreactive spherical inclusions: a report of three cases.

Tauopathies are a major group of neurodegenerative proteinopathies characterized by the accumulation of abnormal and hyperphosphorylated tau proteins in the brain. Tau pathology is characterized as 3R (repeat) or 4R predominant or mixed 3R and 4R type. Here we report three cases lacking mutations in the microtubule associated protein tau (MAPT) gene with unusual tau pathology. The age at onset and duration of illness, respectively, were 63 and 20 years (male), 67 and 5 years (female) and 72 and 20 years (female). The clinical presentation was compatible with a diagnosis of progressive supranuclear palsy (PSP) in two subjects and with cognitive decline in all three subjects. Common neuropathological features included neuronal loss in the hippocampus and dentate gyrus associated with spherical basophilic Pick body-like inclusions showing 4R tau immunoreactivity, which was supported by the detection of predominantly 4R tau species by Western blot examination. In addition, accumulation of tau immunoreactive argyrophilic astrocytes in the hippocampus and amygdala and oligodendroglial coiled bodies in the hippocampal white matter were observed. These morphologies appeared in combination with Alzheimer disease-related pathology and subcortical tau pathology compatible with PSP. Together with a single case report in the literature, our observations on these three cases expand the spectrum of previously described tauopathies. We suggest that this tauopathy variant with hippocampal 4R tau immunoreactive spherical inclusions might contribute to the cognitive deficits in the patients reported here. The precise definition of the clinicopathological relevance of these unusual tau pathologies merits further study.

Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo.

The stereotypical distribution of TAR DNA-binding 43 protein (TDP-43) aggregates in frontotemporal lobar degeneration (FTLD-TDP) suggests that pathological TDP-43 spreads throughout the brain via cell-to-cell transmission and correlates with disease progression, but no in vivo experimental data support this hypothesis. We first develop a doxycycline-inducible cell line expressing GFP-tagged cytoplasmic TDP-43 protein (iGFP-NLSm) as a cell-based system to screen and identify seeding activity of human brain-derived pathological TDP-43 isolated from sporadic FTLD-TDP and familial cases with Granulin (FTLD-TDP-GRN) or C9orf72 repeat expansion mutations (FTLD-TDP-C9+). We demonstrate that intracerebral injections of biologically active pathogenic FTLD-TDP seeds into transgenic mice expressing cytoplasmic human TDP-43 (lines CamKIIa-hTDP-43NLSm, rNLS8, and CamKIIa-208) and non-transgenic mice led to the induction of de-novo TDP-43 pathology. Moreover, TDP-43 pathology progressively spreads throughout the brain in a time-dependent manner via the neuroanatomic connectome. Our study suggests that the progression of FTLD-TDP reflects the templated cell-to-cell transneuronal spread of pathological TDP-43.

TDP-43-positive white matter pathology in frontotemporal lobar degeneration with ubiquitin-positive inclusions.

TDP-43 was recently identified as the major disease protein in neuronal inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). TDP-43 is not only linked to disease mechanisms in FTLD-U, but it is also the most robust marker for the specific detection of neuronal inclusions in FTLD-U. In this study, we describe additional TDP-43 pathology in the white matter as a characteristic feature in a series of 38 FTLD-U cases including 3 cases with mutations in the progranulin gene. White matter pathology was most abundant in frontal and temporal lobes, but it was also detectable in brainstem and spinal cord. Based on morphology and double-labeling experiments, white matter cells with TDP-43-positive inclusions most likely represent oligodendrocytes. Biochemically, hyperphosphorylated and truncated TDP-43 was detectable in insoluble brain extracts from affected white matter regions in FTLD-U, similar to the biochemical signature observed in FTLD-U gray matter. Taken together, these results expand the spectrum of TDP-43 pathology in FTLD-U, suggesting that white matter pathology might contribute to the neurodegenerative process and clinical symptoms in FTLD-U.

Clinical, genetic, and pathologic characteristics of patients with frontotemporal dementia and progranulin mutations.

BACKGROUND: Patients with frontotemporal dementia due to mutation of progranulin may have a distinct phenotype. OBJECTIVE: To identify distinct clinical and pathologic features of patients with frontotemporal dementia who have mutations of progranulin (GRN). DESIGN: Retrospective clinical-pathologic study. SETTING: Academic medical center. PATIENTS: Twenty-eight patients with frontotemporal dementia, including 9 with GRN mutations (4 autopsy cases and 5 with only clinical information) and 19 with the identical pathologic diagnosis--frontotemporal lobar degeneration with ubiquitin-positive and tau-negative inclusions (FTLD-U)--and no GRN mutations. MAIN OUTCOME MEASURES: Demographic, symptom, neuropsychological, and autopsy characteristics. RESULTS: Patients with and without a GRN mutation have similar demographic features, although family history is significantly more common in patients with frontotemporal dementia and a GRN mutation. Both patient groups have frequent social and personality complaints. Neuropsychological evaluation reveals a significant recognition memory deficit in patients with a GRN mutation but a significant language deficit only in patients without a GRN mutation. At autopsy, the semiquantitative burden of ubiquitin abnormality is relatively modest in both groups of patients. CONCLUSION: Patients with a GRN mutation differ clinically from those with the same pathologic diagnosis but no GRN mutation.

Neuron loss and degeneration in the progression of TDP-43 in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is associated with the accumulation of pathological neuronal and glial intracytoplasmic inclusions as well as accompanying neuron loss. We explored if cortical neurons detected by NeuN decreased with increasing TDP-43 inclusion pathology in the postmortem brains of 63 patients with sporadic and familial FTLD-TDP. Semi-automated quantitative algorithms to quantify histology in tissue sections stained with antibodies specific for pathological or phosphorylated TDP-43 (pTDP-43) and NeuN were developed and validated in affected (cerebral cortex) and minimally affected (cerebellar cortex) brain regions of FTLD-TDP cases. Immunohistochemistry (IHC) for NeuN and other neuronal markers found numerous neurons lacking reactivity, suggesting NeuN may reflect neuron health rather than neuron loss in FTLD. We found three patterns of NeuN and pTDP-43 reactivity in our sample of cortical tissue representing three intracortical region-specific stages of FTLD-TDP progression: Group 1 showed low levels of pathological pTDP-43 and high levels NeuN, while Group 2 showed increased levels of pTDP-43, and Group 3 tissues were characterized by reduced staining for both pTDP-43 and NeuN. Comparison of non-C9orf72/GRN FTLD-TDP with cases linked to both GRN mutations and C9orf72 expansions showed a significantly increased frequency of Group 3 histopathology in the latter cases, suggesting more advanced cortical disease. Hence, we propose that IHC profiles of pTDP-43 and NeuN reflect the burden of pTDP-43 and its deleterious effects on neuron health.

Progression of motor neuron disease is accelerated and the ability to recover is compromised with advanced age in rNLS8 mice.

In order to treat progressive paralysis in ALS patients, it is critical to develop a mouse that closely models human ALS in both pathology and also in the timing of these events. We have recently generated new TDP-43 bigenic mice (called rNLS8) with doxycycline (Dox)-suppressible expression of human TDP-43 (hTDP-43) harboring a defective nuclear localization signal (hTDP-43∆NLS) under the control of the NEFH promoter. Our previous studies characterized the pathology and disease course in young rNLS8 mice following induction of neuronal hTDP-43ΔNLS. We now seek to examine if the order and timing of pathologic events are changed in aged mice. We found that the expression of hTDP-43∆NLS in 12+ month old mice did not accelerate the appearance of neuromuscular abnormalities or motor neuron (MN) death in the lumbar spinal cord (SC), though disease progression was accelerated. However, following suppression of the transgene, important differences between young and aged rNLS8 mice emerged in functional motor recovery. We found that recovery was incomplete in aged mice relative to their younger treatment matched counterparts based on gross behavioral measures and physiological recordings from the animals' gastrocnemius (GC) muscles, despite muscle reinnervation by surviving MNs. This is likely because the reinnervation most often only resulted in partial nerve and endplate connections and the muscle's junctional folds were much more disorganized in aged rNLS8 mice. We believe that these studies will be an important basis for the future design and evaluation of therapies designed to slow denervation and promote re-innervation in adult ALS patients.